CN102477446B - Preparation method of promoter for producing ceramide and/or glucosylceramide - Google Patents
Preparation method of promoter for producing ceramide and/or glucosylceramide Download PDFInfo
- Publication number
- CN102477446B CN102477446B CN201110289092.6A CN201110289092A CN102477446B CN 102477446 B CN102477446 B CN 102477446B CN 201110289092 A CN201110289092 A CN 201110289092A CN 102477446 B CN102477446 B CN 102477446B
- Authority
- CN
- China
- Prior art keywords
- ceramide
- glucose
- production accelerant
- debaryomyces
- bean dregs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a preparation method of a promoter for producing ceramide and/or glucosylceramide, wherein debaryomyces microbes are cultured in a culture medium containing bean dregs and saccharides, and the promoter for producing ceramide and/or glucosylceramide is obtained from the supernatant. According to the preparation method of the invention, special microbes are used, and bean dregs which are by products in food processing industry are used as a main raw material of the culture medium; therefore the promoter for producing ceramide and/or glucosylceramide is obtained with low cost and high efficiency.
Description
Technical field
The present invention relates to utilize specified microorganisms, using the by product bean dregs of food-processing industry etc. as the main raw material of substratum, and manufacture can promote skin to produce ceramide and/or the ceramide of glucose ceramide and/or the method for glucose ceramide production accelerant.
Background technology
The ceramide of one of sphingolipid (Ceramide) is present in keratoderma, accounts for 50% left and right of intercellular lipid in stratum corneum.Sphingolipid (Sphingolipid) is the general name with the compound lipid of sphingosine skeleton.Glucose ceramide (Glucosylceramide) is on ceramide, to combine a kind of sphingolipid that glucose forms.By the synthetic ceramide obtaining of epidermic cell, temporarily with the form of glucose ceramide or sphingomyelin (Sphingomyelin), be stored; Be discharged to afterwards extracellular, under the effect of glucose cerebrosidase (Glucocerebrosidase) and sphingomyelinase (Sphingomyelinase), again become ceramide, thereby performance is as the function of intercellular lipid.
All the time, the sphingolipid such as known ceramide, glucose ceramide, galactosyl ceramide has the cuticular maintenance moisture ability of promotion, improves pachylosis texts (patent documentation 1～2).Although skin has the ability that produces ceramide and glucose ceramide, this ability is also insufficient, thereby people have also attempted from the method for external complement ceramide.But the long-term effect of the method can not get approval, and the problem such as existence and stability is low.For example, reported the method (patent documentation 3) of utilizing candida tropicalis (Candida tropicalis) JPCCY0004 strain (NITEP-570) to manufacture glucose ceramide.But the method is not the method about ceramide and/or glucose ceramide production accelerant, and only by the method that in the thalline after cultivating, a large amount of glucose ceramides that exist directly extract; And, by the method, synthesizing the ceramide skeleton of the glucose ceramide obtaining different with the ceramide skeleton of the ceramide type existing in human body, it has carbon-carbon double bond and has side chain at Liang Chu.Thereby, this glucose ceramide is coated on human body skin, likely can not adapt with human body skin and produce the problem of safety in utilization aspect.
And on the other hand, if can strengthen or promote the generation ability of ceramide or the glucose ceramide of skin itself, without from external complement ceramide or glucose ceramide, and can be by promoting skin self to produce barrier function and moisture-keeping functions that ceramide or glucose ceramide improve skin.Like this, because being produces ceramide or glucose ceramide by skin self, thereby there is not the problem of the security aspects such as adaptability with skin is bad in it.
Therefore, in recent years, quite popular about studying with the production that promotes epidermis ceramide to be produced as the material of object in keratoderma.As the material that promotes that ceramide produces, having of having reported be take the ceramide production accelerant (patent documentation 4～6) that the extract of glossy ganoderma, bee pollen (Bee Pollen), Ligusticum wallichii etc. is effective constituent.Yet because these ceramide production accelerants are all to extract and to obtain from plant material, this extractions operates the length that expends time in, and due to the reasons such as possibility is limited of utilizing of natural resource, its manufacturing cost is high.
In addition, also reported and take the ceramide synthesis accelerators (patent documentation 7) that the lactic acid bacteria culture that contains nicotinic acid and/or niacinamide or Yeast culture be effective constituent.Yet, from the embodiment of patent documentation 7, can find out, do not contain the lactic acid bacteria culture of nicotinic acid and/or niacinamide or the ceramide generation of Yeast culture (comparative example 1～4) there is no considerable change than control group, thereby this ceramide synthesis accelerators contains in fact nicotinic acid and/or niacinamide as effective constituent, the synthetic facilitation effect of its ceramide is abundant not enough, the leeway being still improved.And owing to using nicotinic acid or niacinamide, thereby its manufacturing cost is higher.
In addition, the microorganism of known Debaryomyces (Debaryomyces) generally has following industrial use, and for example, glucose fermentation is produced D-R alcohol or wood sugar is changed into Xylitol.
But, also do not utilize at present the microorganism of Debaryomyces (Debaryomyces) to manufacture the report of ceramide and/or glucose ceramide production accelerant.
Patent documentation 1: Japanese kokai publication sho 61-260008 communique
Patent documentation 2: Japanese kokai publication sho 61-271205 communique
Patent documentation 3: TOHKEMY 2005-194240 communique
Patent documentation 4: TOHKEMY 2010-70499 communique
Patent documentation 5: TOHKEMY 2010-150237 communique
Patent documentation 6: Japanese kokai publication hei 9-194383 communique
Patent documentation 7: TOHKEMY 2010-22217 communique
Summary of the invention
For the technical problem existing in above-mentioned prior art, the inventor has carried out concentrated research, found that: the microorganism of cultivating Debaryomyces (Debaryomyces) in the substratum that contains bean dregs and carbohydrate, particularly cultivate the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) or the beautiful Dbaly yeast of model (Debaryomycesvarrijiae), can be low-cost and obtain efficiently ceramide and/or glucose ceramide production accelerant, thus the present invention completed.
The present invention relates to, the manufacture method of a kind of ceramide and/or glucose ceramide production accelerant is provided, wherein, in the substratum that contains bean dregs and carbohydrate, cultivate the microorganism of Debaryomyces (Debaryomyces), in clear liquid, obtain from it ceramide and/or glucose ceramide production accelerant.
The invention still further relates to, provide the fermented product extract of Debaryomyces (Debaryomyces) of bean dregs for promoting the purposes of non-therapeutic purpose of the generation of ceramide and/or glucose ceramide.
The invention still further relates to, provide the fermented product extract of Debaryomyces (Debaryomyces) of bean dregs as the purposes of the non-therapeutic purpose of ceramide and/or glucose ceramide production accelerant.
The invention still further relates to the purposes of the fermented product extract of Debaryomyces (Debaryomyces) that bean dregs are provided in manufacturing ceramide and/or glucose ceramide production accelerant.
Invention effect
Manufacturing method according to the invention, can utilize specified microorganisms, using the by product bean dregs of food-processing industry etc. as the main raw material of substratum, low-cost and obtain efficiently ceramide and/or glucose ceramide production accelerant, this promotor can effectively be improved the ability of generation ceramide/glucose ceramide that skin has, thereby effectively improves the moisture-retaining capacity of skin.
Embodiment
(microorganism that can manufacture for ceramide and/or glucose ceramide production accelerant)
As the microorganism of the Debaryomyces (Debaryomyces) using in the present invention, can list at least a kind of microorganism selecting the inferior Dbaly yeast of the Chinese (Debaryomyceshansenii) that uses from field of food, multiform Dbaly yeast (Debaryomyces polymorphus), the beautiful Dbaly yeast of model (Debaryomyces vanrijiae) etc.; The variant that also comprises these microorganisms that obtain according to usual method.These microorganisms are as described below to play a role, by cultivating in the substratum containing bean dregs and carbohydrate, thus the material that the ceramide of the epidermic cell that can be improved in clear liquid from it and/or glucose ceramide produce ability.Wherein, from obtained product, there is the viewpoint that higher ceramide produces facilitation effect and glucose ceramide generation facilitation effect, the preferably inferior Dbaly yeast of the Chinese (Debaryomyceshansenii) or the beautiful Dbaly yeast of model (Debaryomyces vanrijiae).
The microorganism of Debaryomyces involved in the present invention (Debaryomyces) can be bought and obtain from commercial channel such as Chinese common micro-organisms preservation administrative center (CGMCC), Chinese Typical Representative culture collection center (CCTCC), Chinese Universities ' industrial microorganism resource and information centers (CICIM:Culture & Information Center of Industrial Microorganism of China Universities).
(manufacture method of ceramide and/or glucose ceramide production accelerant)
The manufacture method of ceramide of the present invention and/or glucose ceramide production accelerant, by the substratum containing bean dregs and carbohydrate, is cultivated mentioned microorganism, and in clear liquid, is obtained ceramide and/or glucose ceramide production accelerant from it.The concrete steps of the method are as follows.
< substratum >
In the present invention, preferably cultivate one kind or two or more in mentioned microorganism in liquid medium within.
In the present invention, the main component of liquid nutrient medium is bean dregs and carbohydrate.Also can suitably add peptone, yeast powder.
Because bean dregs and carbohydrate are all the materials easily obtaining, thereby substratum of the present invention has advantages of that cost is low.
Wherein, bean dregs refer to, soybeans soaking are made in water it fully absorb after moisture, then through pulverizing, remove by filter water and the material that obtains; As mentioned above, especially can use and in food industries, squeeze remaining bean dregs after soya-bean milk.
As carbohydrate, can use separately any one in glucose, maltose, sucrose etc., or mix and use.From improving the viewpoint of fermentative production and usability, preferred glucose wherein.
In the present invention, in order to improve, ceramide produces facilitation effect and/or glucose ceramide produces facilitation effect, medium optimization of the present invention consists of bean dregs, glucose and water, further the content of the bean dregs in preferred culture medium is 5～20w/v%, 8～12w/v% more preferably, and the content of glucose is 0.2～2w/v%, 0.8～1.2w/v% more preferably.
Below, the above-mentioned this substratum for the manufacture of ceramide and/or glucose ceramide production accelerant is called to " ceramide/glucose ceramide production accelerant is manufactured substratum ".
< culture condition >
The thalline of mentioned microorganism is directly inoculated into ceramide/glucose ceramide production accelerant to be manufactured in substratum.As the culture condition for the manufacture of ceramide and/or glucose ceramide production accelerant, from the necessary temperature of growth, the time of optimization Debaryomyces (Debaryomyces) microorganism, and the viewpoint that improves ceramide generation facilitation effect and glucose ceramide generation facilitation effect is set out, preferably 20～40 ℃, more preferably under the culture temperature of 27～35 ℃, cultivate 1～7 day, preferably cultivate 48～96 hours, more preferably cultivate 68～76 hours.
The recovery of < ceramide and/or glucose ceramide production accelerant, refining >
The nutrient solution obtaining from above-mentioned cultivation, reclaim, refine ceramide and/or glucose ceramide production accelerant, can as required, suitably combine the common operations such as filtration, centrifugation, ion-exchange or adsorption chromatography, solvent extraction, crystallization.Particularly, for example, can from nutrient solution, remove by centrifugation thalline, then pulverize in ultrasonic treatment unit, reclaim the supernatant liquor after centrifugal.Then, then the supernatant liquid filtering after reclaiming is removed to impurity etc., under vacuum, after drying treatment, obtain fermenting bean dregs extract as ceramide of the present invention and/or glucose ceramide production accelerant.
As shown in following embodiment, by cultivating Debaryomyces of the present invention (Debaryomyces) microorganism, and the fermenting bean dregs extract obtaining from culture supernatant, in normal human's keratinocyte, there is the effect of the amount increase that makes ceramide and/or glucose ceramide.
Therefore, by the fermenting bean dregs extract of cultivating Debaryomyces of the present invention (Debaryomyces) microorganism and obtaining from culture supernatant, can be for promoting ceramide and/or the therapeutic purpose of glucose ceramide generation or the use of non-therapeutic purpose, the ceramide and/or the glucose ceramide production accelerant that also can be used as therapeutic purpose or non-therapeutic purpose are used.The use of non-therapeutic purpose is that to improve looks be the use of object, or for maintaining the use etc. of state of health.And the use of described therapeutic purpose and the use of non-therapeutic purpose can completely be carried out with distinguishing.
In addition, by the fermenting bean dregs extract of cultivating Debaryomyces of the present invention (Debaryomyces) microorganism and obtaining from culture supernatant, can also be for the manufacture of ceramide and/or glucose ceramide production accelerant.
Ceramide of the present invention and/or glucose ceramide production accelerant can be used as the ceramide that makes in stratum corneum and/or glucose ceramide increases, and recovers or maintain the uses such as the barrier function of skin and the pharmaceuticals of moisture-keeping functions, accurate pharmaceuticals, makeup.In addition, ceramide production accelerant of the present invention or glucose ceramide production accelerant also can be used as take that ceramide produce to promote or glucose ceramide produce promote be concept, and indicate that as required the accurate pharmaceuticals of this concept, makeup are used.
Use form as ceramide of the present invention and/or glucose ceramide production accelerant, can be used as the additive adding in culturing cell, or make the external composition for skin such as detergent, makeup, according to using method, can provide with forms such as astringent, emulsion, gel, frost, ointment, powder, particles.When the various external composition for skin of preparation, can use individually ceramide of the present invention and/or glucose ceramide production accelerant, or the use such as the appropriately combined oiliness composition conventionally coordinating in the external composition for skin such as detergent and makeup, wetting Agent for Printing Inks, powder, pigment, emulsifying agent, solubilizing agent, UV light absorber, thickening material, effective component, spices, fungi-proofing mould inhibitor, plant milk extract, alcohols.
In the situation that promote ceramide and/or glucose ceramide in ceramide of the present invention and/or glucose ceramide production accelerant being added to the epidermic cell of cultivation, as fermenting bean dregs extract of the present invention (being scaled drying solid composition), its addition is preferably 0.0001～10w/v%, more preferably 0.001～5w/v%.When addition is greater than 0.0001w/v%, can obtain sufficient facilitation effect; And when addition is less than 10w/v%, less to the pungency of epidermic cell.
When ceramide of the present invention and/or glucose ceramide production accelerant being engaged in to external composition for skin and using, thereby effectively improve the moisture-retaining capacity of skin and be difficult for demonstrating the color of culture and the viewpoint of smell from the generation of effective promotion ceramide and/or glucose ceramide, drying solid composition as fermenting bean dregs extract, its use level is preferably 0.01～20w/v% of external composition for skin total amount, more preferably 0.1～10w/v%.
Embodiment
Below, based on embodiment, the present invention will be described in more detail, but the present invention is not limited thereto.
Embodiment 1
(manufacture of ceramide and/or glucose ceramide production accelerant)
As manufacture the microorganism of ceramide and/or glucose ceramide production accelerant from bean dregs, use the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) (from Southern Yangtze University, to buy, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, original number 476-20-1).
The above-mentioned bacterial strains obtaining after cultivating at 30 ℃ in YPD (Yeast Extract Peptone Dextrose Medium) substratum is inoculated in the Erlenmeyer flask (capacity 300ml) that contains ceramide/glucose ceramide production accelerant manufacture substratum 100ml, at 30 ℃, cultivates 72 hours.This substratum contains 10w/v% bean dregs and 1w/v% glucose.Wherein, the preparation of bean dregs is soaked 12 hours by soybean being put into water, makes soybean fully absorb moisture, then the soybean that has absorbed moisture is ground to fragmentation, filters and removes after liquid component, and residual residue is drained to obtain.
Then, in order to reclaim ceramide and/or glucose ceramide production accelerant the nutrient solution from obtaining, with 3000rpm centrifugation, within 10 minutes, remove thalline, reclaim the supernatant liquor after centrifugation, in culture supernatant after recovery, add the dehydrated alcohol of 2.5 times, use again ultrasonic treatment unit (manufacture of Branson company) to pulverize 10 minutes, with 3000rpm centrifugation 30 minutes, reclaim supernatant liquor.With the about 70ml of this supernatant liquor of filter paper filtering, drier this filtered liquid, to constant weight, obtains fermenting bean dregs extract, as ceramide/glucose ceramide production accelerant of the present invention in Vacuumdrier below 40 ℃.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 1w/v%, be kept at 4 ℃, for ceramide/glucose ceramide afterwards, produce and promote test.
Embodiment 2
As manufacture the microorganism of ceramide/glucose ceramide production accelerant from bean dregs, use the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) (from Southern Yangtze University, to buy, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, original number 514-20-4).Adopt the method identical with embodiment 1 to obtain fermenting bean dregs extract, as ceramide/glucose ceramide production accelerant of the present invention.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 1w/v%, be kept at 4 ℃, for ceramide/glucose ceramide afterwards, produce and promote test.
Embodiment 3
As manufacture the microorganism of ceramide/glucose ceramide production accelerant from bean dregs, use the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) (from Southern Yangtze University, to buy, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0356).Adopt the method identical with embodiment 1 to obtain fermenting bean dregs extract, as ceramide/glucose ceramide production accelerant of the present invention.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 1w/v%, be kept at 4 ℃, for ceramide/glucose ceramide afterwards, produce and promote test.
Embodiment 4
As manufacture the microorganism of ceramide/glucose ceramide production accelerant from bean dregs, use the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) (from Southern Yangtze University, to buy, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0286).Adopt the method identical with embodiment 1 to obtain fermenting bean dregs extract, as ceramide/glucose ceramide production accelerant of the present invention.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 1w/v%, be kept at 4 ℃, for ceramide/glucose ceramide afterwards, produce and promote test.
Embodiment 5
As manufacture the microorganism of ceramide/glucose ceramide production accelerant from bean dregs, use the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) (from Southern Yangtze University, to buy, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0287).Adopt the method identical with embodiment 1 to obtain fermenting bean dregs extract, as ceramide/glucose ceramide production accelerant of the present invention.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 1w/v%, be kept at 4 ℃, for ceramide/glucose ceramide afterwards, produce and promote test.
Embodiment 6
As manufacture the microorganism of ceramide/glucose ceramide production accelerant from bean dregs, use the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) (from Southern Yangtze University, to buy, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0289).Adopt the method identical with embodiment 1 to obtain fermenting bean dregs extract, as ceramide/glucose ceramide production accelerant of the present invention.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 1w/v%, be kept at 4 ℃, for ceramide/glucose ceramide afterwards, produce and promote test.
Embodiment 7
As manufacture the microorganism of ceramide/glucose ceramide production accelerant from bean dregs, use the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) (from Southern Yangtze University, to buy, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0290).Adopt the method identical with embodiment 1 to obtain fermenting bean dregs extract, as ceramide/glucose ceramide production accelerant of the present invention.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 1w/v%, be kept at 4 ℃, for ceramide/glucose ceramide afterwards, produce and promote test.
Embodiment 8
As manufacture the microorganism of ceramide/glucose ceramide production accelerant from bean dregs, use the beautiful Dbaly yeast of model (Debaryomyces vanrijiae) (from Southern Yangtze University, to buy, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0198).Adopt the method identical with embodiment 1 to obtain fermenting bean dregs extract, as ceramide/glucose ceramide production accelerant of the present invention.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 0.1w/v%, be kept at 4 ℃, for ceramide/glucose ceramide afterwards, produce and promote test.
Embodiment 9
As manufacture the microorganism of ceramide/glucose ceramide production accelerant from bean dregs, use the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) (from Southern Yangtze University, to buy, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0351).Adopt the method identical with embodiment 1 to obtain fermenting bean dregs extract, as ceramide/glucose ceramide production accelerant of the present invention.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 1w/v%, be kept at 4 ℃, for ceramide/glucose ceramide afterwards, produce and promote test.
(ceramide/glucose ceramide produces and promotes test)
< keratinocyte (Keratinocytes) is cultivated >
First, activation keratinocyte (Cascade Co., Ltd) is as primary cell, in 75cm
2in culturing bottle (the substratum 2ml that contains somatomedin), at 37 ℃, CO
2concentration is to carry out cell cultures under 5% condition, until cultivate, to cell aggregation density (confluence), is 80～90%.Passage cell is to 175cm again
2in square vase, continue culturing cell to 80～90% concentration.Then with 1 * 10
5individual cell/ml is passaged in 12 orifice plates (every pore volume is 2ml), continues culturing cell to 80～90% concentration.
Then, change the substratum that does not contain somatomedin, in above-mentioned 12 orifice plates, (n=2) adds the aqueous ethanolic solution containing 1w/v% fermenting bean dregs extract in above-described embodiment 1～9 with the amount in 20 μ l/ holes respectively.In addition, only add 50v/v% ethanol (not adding ceramide/glucose ceramide production accelerant) product in contrast, under same culture conditions, cultivate 72 hours.Cultivate after 72 hours, supernatant discarded nutrient solution, with 2ml PBS (phosphate buffered saline buffer), clean twice, cell (2ml/ hole), add again 1ml PBS to plate hole, with cell harvestor (cell shovel) collecting cell, to test tube, prepare against follow-up ceramide/glucose ceramide and protein analysis.
< lipids extraction >
Lipids extraction adopts Bligh & Dyer method to carry out.Particularly, first, in the celliferous PBS liquid obtaining in above-mentioned recovery, add the solvent of methyl alcohol: chloroform=2.5ml: 1.25ml, vibrate 20 minutes, carry out centrifugation (3000rpm, 5 minutes), thereby obtain supernatant liquor and precipitation.Supernatant A is transferred in other test tube for the quantitative analysis of ceramide/glucose ceramide to the analysis by deposit B for protein content.
< protein content is analyzed >
For the ceramide/glucose ceramide amount in characterize cells, carry out protein content analysis.In the deposit B of above-mentioned recovery, add SDS (sodium lauryl sulphate) solution of 900 μ l and mix, under 60 ℃ of water-baths, heat and within 2 hours, make protein denaturation degraded, after cooling, add therein the 2N HCl solution of 100 μ l, according to BCA Kit (BCA determination of protein concentration test kit) method, measure the wherein amount of protein (be designated as Pro, unit is μ g/ml).
< ceramide/glucose ceramide component analysis >
The solvent that adds PBS: chloroform=1.25ml: 1.25ml in residual supernatant A; vibrate 20 minutes; 3000rpm carries out centrifugation 5 minutes; separated, reclaim the chloroform layer as lower phase; and dry chloroform layer obtains dry product for 40 minutes under 30 ℃, nitrogen protection; the solvent of the chloroform that adds 100 μ l: methyl alcohol=2.5ml: 1.25ml; and by following thin plate chromatography (TLC) method, the amount of ceramide and glucose ceramide (be designated as respectively Cer and GlyCer, unit is μ g/ml) is carried out quantitatively.
Adopt dry TLC plate, take chloroform: methyl alcohol: acetic acid (190: 9: 1v/v/v) as developing agent carries out thin plate chromatography, treat that developing agent walks to thin plate top, dries up developing agent with blower; Repeat above step once.Then take chloroform: methyl alcohol: acetone (76: 20: 4v/v/v) as developing agent carries out thin plate chromatography, treat that developing agent walks to apart from 2.5cm place, thin plate bottom, stop exhibition layer, dry up with blower.Spray, with copper sulfate phosphoric acid solution, dries up, and chromatoplate is positioned over and in 180 ℃ of bake plate, toasts colour developing in 7 minutes.Scanning colour developing TLC plate also carries out ceramide (or glucose ceramide) analysis.
Then, according to following formula (1), income value, respectively divided by protein content separately, is obtained to Cer/Pro and GlyCer/Pro.Cer/Pro and the GlyCer/Pro of reference substance of take is 1, obtains the relative value of Cer/Pro of the present invention and GlyCer/Pro.And the amount of the protein in the every hole of the reference substance of take is 100, obtain ceramide in every hole and the amount (being designated as respectively Cer/Well and GlyCer/Well, with respect to the relative quantity of reference substance) of glucose ceramide.In table 1, represent in the lump.
Formula (1)
Cer/Pro=ceramide amount (μ g/ml)/protein content (μ g/ml)
GlyCer/Pro=glucose ceramide amount (μ g/ml)/protein content (μ g/ml)
Table 1
| Cer/Pro | Cer/Well | GlyCer/Pro | GlyCer/Well | |
| Reference substance | 1 | 1 | 1 | 1 |
| Embodiment 1 | 2.68 | 3.17 | 2.29 | 2.70 |
| Embodiment 2 | 2.57 | 3.02 | 0.61 | 0.71 |
| Embodiment 3 | 0.09 | 0.12 | 1.32 | 1.66 |
| Embodiment 4 | 3.77 | 4.99 | 0.36 | 0.48 |
| Embodiment 5 | 3.05 | 3.71 | 0.20 | 0.25 |
| Embodiment 6 | 1.55 | 1.46 | 0.34 | 0.32 |
| Embodiment 7 | 1.03 | 1.38 | 0.07 | 0.10 |
| Embodiment 8 | 0.99 | 0.91 | 1.13 | 1.04 |
| Embodiment 9 | 1.53 | 1.40 | 1.35 | 1.23 |
As shown in table 1, the product of the present invention that obtain in embodiment 1 and embodiment 9 had both had ceramide and had produced facilitation effect, had again glucose ceramide and produced facilitation effect; In addition, it is remarkable that the ceramide of the product of the present invention that obtain in embodiment 2 and embodiment 4～7 produces facilitation effect, and the glucose ceramide of the product of the present invention that obtain in embodiment 3 and embodiment 8 generation facilitation effect is remarkable.Therefore, confirmed that ceramide/glucose ceramide production accelerant of the present invention has ceramide and produces facilitation effect and/or glucose ceramide generation facilitation effect.
(in ceramide/glucose ceramide production accelerant of the present invention, there is not the test of ceramide and glucose ceramide)
Without cell cultures, directly adopt TLC method to analyze the ceramide/glucose ceramide production accelerant obtaining in above-described embodiment 1～9, result shows in product of the present invention, there is no ceramide and glucose ceramide.
Show thus, although there is not ceramide or glucose ceramide in product of the present invention itself, but have, ceramide produces facilitation effect and/or glucose ceramide produces facilitation effect, can be used as ceramide and/or glucose ceramide production accelerant and uses.
Claims (8)
1. a manufacture method for ceramide and/or glucose ceramide production accelerant, wherein,
In the substratum that contains bean dregs and carbohydrate, cultivate the microorganism of Debaryomyces (Debaryomyces), in clear liquid, obtain from it ceramide and/or glucose ceramide production accelerant,
The microorganism of described Debaryomyces (Debaryomyces) is the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) or the beautiful Dbaly yeast of model (Debaryomyces vanrijiae).
2. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
Described carbohydrate is glucose.
3. the manufacture method of ceramide as claimed in claim 2 and/or glucose ceramide production accelerant, wherein,
Described substratum consists of bean dregs, glucose and water.
4. the manufacture method of ceramide as claimed in claim 3 and/or glucose ceramide production accelerant, wherein,
The content of bean dregs described in described substratum is 8～12w/v%, and the content of described carbohydrate is 0.8～1.2w/v%.
5. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
Described culture temperature is 27～35 ℃.
6. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
Described incubation time is 1～7 day.
7. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
The nutrient solution obtaining by described cultivation is carried out centrifugal, thereby obtain described supernatant liquor.
8. the manufacture method of ceramide as claimed in claim 7 and/or glucose ceramide production accelerant, wherein,
In the described supernatant liquor of centrifugal rear acquisition, add after ethanol, further, after ultrasonication, centrifugation, filtration, vacuum drying treatment are refined, obtain ceramide and/or glucose ceramide production accelerant.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110289092.6A CN102477446B (en) | 2010-11-25 | 2011-09-15 | Preparation method of promoter for producing ceramide and/or glucosylceramide |
| JP2013522070A JP5639713B2 (en) | 2010-11-09 | 2011-11-08 | Method for producing ceramide and / or glucosylceramide production promoter |
| PCT/CN2011/001877 WO2012062043A1 (en) | 2010-11-09 | 2011-11-08 | Method for producing promoter from ceramide and/or glucosylceramide |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010570389 | 2010-11-25 | ||
| CN201010570389.5 | 2010-11-25 | ||
| CN201110289092.6A CN102477446B (en) | 2010-11-25 | 2011-09-15 | Preparation method of promoter for producing ceramide and/or glucosylceramide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102477446A CN102477446A (en) | 2012-05-30 |
| CN102477446B true CN102477446B (en) | 2014-03-19 |
Family
ID=46090230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110289092.6A Expired - Fee Related CN102477446B (en) | 2010-11-09 | 2011-09-15 | Preparation method of promoter for producing ceramide and/or glucosylceramide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102477446B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101171340A (en) * | 2005-04-13 | 2008-04-30 | 泰特&莱尔组分美国公司 | Ascorbic acid production from D-glucose in yeast |
| WO2010013179A1 (en) * | 2008-07-29 | 2010-02-04 | L'oreal | Cosmetic use of microorganisms for the treatment of oily skin |
| CN101653561A (en) * | 2008-08-18 | 2010-02-24 | 复旦大学附属中山医院 | Ceramide production accelerant |
| CN101705193A (en) * | 2009-11-20 | 2010-05-12 | 中国水产科学研究院黄海水产研究所 | Astaxanthin-producing ocean rhodotorula YS-185 and method for producing astaxanthin thereof |
-
2011
- 2011-09-15 CN CN201110289092.6A patent/CN102477446B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101171340A (en) * | 2005-04-13 | 2008-04-30 | 泰特&莱尔组分美国公司 | Ascorbic acid production from D-glucose in yeast |
| WO2010013179A1 (en) * | 2008-07-29 | 2010-02-04 | L'oreal | Cosmetic use of microorganisms for the treatment of oily skin |
| CN101653561A (en) * | 2008-08-18 | 2010-02-24 | 复旦大学附属中山医院 | Ceramide production accelerant |
| CN101705193A (en) * | 2009-11-20 | 2010-05-12 | 中国水产科学研究院黄海水产研究所 | Astaxanthin-producing ocean rhodotorula YS-185 and method for producing astaxanthin thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102477446A (en) | 2012-05-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114209617B (en) | Ganoderma lucidum extract fermented by yeast and preparation method and application thereof | |
| CN103689550A (en) | Ganoderma lucidum functional food and preparation method thereof | |
| CN105062677A (en) | Method for preparing fermented beet extract through processing beet juice by Lactobacillus plantarum | |
| CN103393584A (en) | Cucumber moisturizing liquid | |
| CN102488087A (en) | Biological detoxification method for camellia seed cakes | |
| CN103451108B (en) | Aureobasidium pullulans OF-01 strain and method for preparing tobacco endogenous derived spice by using strain | |
| CN103060385B (en) | Preparation method for producing promoter by ceramide and/or glucosylceramide | |
| CN102477446B (en) | Preparation method of promoter for producing ceramide and/or glucosylceramide | |
| KR20130112401A (en) | Method for manufacturing ferment extract from rice bran and cosmetic compositions containing threrof | |
| CN106636252A (en) | Thelephora ganbajun Zang exopolysaccharide, preparation method thereof and application of exopolysaccharide | |
| CN102994556B (en) | Manufacturing method of ceramide and/or glucose ceramide production promoter | |
| CN102465151A (en) | Manufacturing method of ceramide generation accelerator and/or glucosylceramide generation accelerator | |
| CN102994555B (en) | Manufacturing method of ceramide and/or glucose ceramide production promoter | |
| CN103060384B (en) | Making method of ceramide and/or glucosylceramide generation accelerator | |
| CN102994554A (en) | Manufacturing method of ceramide and/or glucose ceramide production promoter | |
| CN103060383B (en) | Ceramide and/or glucose ceramide produce the manufacture method of promotor | |
| CN102465150B (en) | Manufacturing method of ceramide generation accelerator | |
| CN103087887A (en) | Purple sweet potato health care wine and preparation method thereof | |
| CN103060382B (en) | The manufacture method of ceramide production accelerant | |
| CN102994557B (en) | Manufacturing method of ceramide production promoter | |
| JP6181914B2 (en) | Ceramide production promoter | |
| CN102994558B (en) | Manufacturing method of ceramide production promoter | |
| CN102827876B (en) | Preparation method of ceramide production accelerator | |
| CN102559767B (en) | Manufacturing method of ceramide production accelerating agent | |
| CN104489203A (en) | Ganoderma and tartary buckwheat compounded fermented tea and producing method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140319 Termination date: 20170915 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |